1. Field of the Invention
The invention relates generally to the fields of autoimmunity and inflammatory bowel disease and more specifically to genetic methods for diagnosing Crohn""s disease and other autoimmune diseases.
2. Background Information
Inflammatory bowel disease (IBD) is the collective term used to describe two gastrointestinal disorders of unknown etiology: Crohn""s disease (CD) and ulcerative colitis (UC). The course and prognosis of IBD, which occurs world-wide and is reported to afflict as many as two million people, varies widely. Onset of IBD is predominantly in young adulthood with diarrhea, abdominal pain, and fever the three most common presenting symptoms. The diarrhea may range from mild to severe, and anemia and weight loss are additional common signs of IBD. Ten percent to fifteen percent of all patients with IBD will require surgery over a ten year period. In addition, patients with IBD are at increased risk for the development of intestinal cancer. Reports of an increasing occurrence of psychological problems, including anxiety and depression, are perhaps not surprising symptoms of what is often a debilitating disease that strikes people in the prime of life.
A battery of laboratory, radiological, and endoscopic evaluations are typically combined to derive a diagnosis of IBD and to assess the extent and severity of the disease. Nevertheless, differentiating Crohn""s disease from ulcerative colitis, as well as other types of inflammatory conditions of the bowel, such as irritable bowel syndrome, infectious diarrhea, rectal bleeding, radiation colitis and the like, is difficult because the mucosa of the small and large intestines reacts in a similar way to a large number of different insults. Furthermore, the extensive and often protracted clinical testing required to diagnose CD delays accurate diagnosis and treatment and involves invasive procedures such as endoscopy.
To date, a reliable genetic test for Crohn""s disease, which would be highly prized as a non-invasive method for the early diagnosis of Crohn""s disease, is not available. Such a test, based on identifying genetic markers that are associated with a predisposing mutation to an autoimmune disease such as Crohn""s disease, would obviate invasive clinical procedures. Such a genetic assay also would be useful in methods of predicting susceptibility to Crohn""s disease in asymptomatic individuals, making prophylactic therapy possible. Unfortunately, genetic markers closely associated with Crohn""s disease have yet to be identified. The present invention satisfies this need and provides related advantages as well.
The present invention provides a method of diagnosing or predicting susceptibility to an autoimmune disease in an individual by determining the presence or absence in the individual of a 2-2-4 haplotype at the Notch4, HSP70-HOM and D6S273 loci, where the presence of the haplotype diagnoses or predicts susceptibility to the autoimmune disease. The methods of the invention can be particularly useful for diagnosing or predicting susceptibility to Crohn""s disease, rheumatoid arthritis and Type I diabetes mellitus. In a preferred embodiment, the methods of the invention are used to diagnose or predict susceptibility to Crohn""s disease in an individual of Ashkenazi Jewish ethnicity.
In one embodiment, the invention provides a method of diagnosing or predicting susceptibility to an autoimmune disease by determining the presence or absence of the 2-2-4 haplotype using enzymatic amplification of nucleic acid from the individual. Subsequent to such enzymatic amplification, the presence or absence of the 2-2-4 haplotype can be determined by electrophoresis such as automated capillary electrophoresis. In another embodiment, the presence or absence of the 2-2-4 haplotype can be determined following enzymatic amplification using restriction fragment length polymorphism analysis. In another embodiment, following enzymatic amplification, the presence or absence of the 2-2-4 haplotype is determined using sequence analysis.
In one embodiment, the presence or absence of the 2-2-4 haplotype can be determined by obtaining material including nucleic acid including Notch4, HSP70-HOM and D6S273 loci from the individual, enzymatically amplifying the nucleic acid to produce a first amplified fragment containing the Notch4 locus, enzymatically amplifying the nucleic acid to produce a second amplified fragment containing the HSP70-HOM locus, and enzymatically amplifying the nucleic acid to produce a third amplified fragment containing the D6S273 locus. Additionally, the presence or absence of the 2-2-4 haplotype can be determined by electrophoresing the first amplified fragment to determine whether a Notch4 allele 2 is present, electrophoresing the second amplified fragment to determine whether a HSP70-HOM allele 2 fragment is present and electrophoresing the third amplified fragment to determine whether a D6S273 allele 4 is present. The presence of the Notch4 allele 2, the HSP70-HOM allele 2 and the D6S273 allele 4 indicates that the 2-2-4 haplotype is present. In one embodiment, the second amplified fragment is restricted with Nco I or an isoschizomer thereof.
The invention also provides a method of diagnosing or predicting susceptibility to an autoimmune disease in an individual by determining the presence or absence in the individual of a disease-associated haplotype which is associated with a 2-2-4 haplotype at the Notch4, HSP70-HOM and D6S273 loci, where the presence of the disease-associated haplotype is diagnostic of or predictive of susceptibility to the autoimmune disease. In one embodiment, the disease-associated haplotype is associated with the autoimmune disease with an odds ratio of at least 5 and a lower 95% confidence limit of greater than 1. The methods of the invention are useful for diagnosing an autoimmune disease such as Crohn""s disease, rheumatoid arthritis or diabetes and can be particularly useful for diagnosing Crohn""s disease in an individual of Ashkenazi Jewish ethnicity.
The present invention also provides methods for diagnosing or predicting susceptibility to an autoimmune disease in an individual by determining the presence or absence of a disease-associated allele associated with the 2-2-4 haplotype at the Notch4, HSP70-HOM and D6S273 loci, where the presence of the disease-associated allele is diagnostic of or predictive of susceptibility to the autoimmune disease. In one embodiment, the disease-associated allele is associated with the autoimmune disease with an odds ratio of at least 5 and a lower 95% confidence limit of greater than 1.